Method for quantitatively analyzing an anticoagulant in a sample

ABSTRACT

A method for quantitatively analyzing an anticoagulant in a sample may include: (a) providing and incubating a reaction mixture comprising (i) the sample, (ii) a defined amount of an activated coagulation factor whose activity is directly or indirectly influenceable by the anticoagulant to be determined, wherein the activated coagulation factor is present in a separate reagent which is added to the reaction mixture, (iii) a cleavable substrate which has at least one cleavage site for the activated coagulation factor, and (iv) a solid phase to which the cleavable substrate is bound or becomes bound during the incubation; (b) separating the solid phase; and (c) determining the amount of solid-phase-bound, uncleaved substrate, wherein the determined amount of the solid-phase-bound uncleaved substrate is proportional to an amount or activity of the anticoagulant in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to EP Patent Application No. 09015240filed Dec. 9, 2009, the contents of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention lies in the field of coagulation diagnostics andrelates to methods for determining the activity of proteolytic bloodcoagulation factors and also to methods for determining anticoagulants,which inhibit the activity of blood coagulation factors.

BACKGROUND

Coagulation diagnostics are divided into global tests for examining thefunctionality of the blood coagulation cascade and individual tests fordetermining the activity of individual blood coagulation factors.Different test formats are known for both global tests and individualtests. Test formats are essentially divided into coagulation tests andchromogenic tests.

In a coagulation test, the patient sample to be examined, which usuallyconsists of plasma, is mixed with a coagulation activator which startsthe coagulation process. The coagulation process leads to the formationof a fibrin clot which can be measured with the aid of photometricmethods. The rate of fibrin formation is a measure of the functionalityof the blood coagulation cascade. To determine the activity of anindividual coagulation factor in a coagulation test, the patient sampleto be examined is additionally mixed with deficient plasma whichsupplies all components of the blood coagulation cascade except theblood coagulation factor to be tested.

In a chromogenic test, the patient sample to be examined, which usuallyconsists of plasma, is mixed with a coagulation activator and with asubstrate for a coagulation factor. Since most blood coagulation factorsare serine endopeptidases, i.e., hydrolases which can cleave peptidebonds, use is predominantly made of peptide substrates which are cleavedhighly specifically by the blood coagulation factor to be determined andwhich have a detectable signaling moiety. It may be preferred to usecleavable chromogenic or fluorogenic signalling moieties which aredetermined photometrically. Patent documents EP 34122 A1 and U.S. Pat.No. 4,508,644 describe a multiplicity of chromogenic peptide substratesand the use thereof in coagulation diagnostic tests, e.g., fordetermining the coagulation factors factor IIa (thrombin) and Xa. Indocument EP 78764 A1, a chromogenic method for determining thecoagulation factor XIIa is described.

Particularly chromogenic tests can also be used to determineanticoagulants, which inhibit the activity of blood coagulation factors,in patient samples. For this purpose, the patient sample to be examinedis mixed with an activated coagulation factor and with a substrate forthis coagulation factor. The more anticoagulant that is present in thesample, the stronger is the inhibition of the activated coagulationfactor and the less substrate that is cleaved.

These known methods are homogeneous methods. Homogeneous test methodshave the advantage that the sample is mixed with the detection reagentsto form a test mix and that the detection reaction is measured in thistest mix without the need for additional separation steps, for example,for separating the analyte from the rest of the sample constituents.Homogeneous test methods, however, also have the disadvantage thatsubstances intrinsic to the sample are present during the entire testmethod and, as a result, can affect the detection reaction or caninterfere with the measurement of the detection reaction. Patientsamples can, for example in individual cases, contain abnormally highconcentrations of one or more intrinsic, i.e., endogenous substances,which can prove disruptive on exceeding a tolerable concentration inphotometric detection methods and can lead to a systematic error. It iswell known that problems are caused by hemolytic, icteric, and/orlipemic serum or plasma samples, so-called HIL samples which haveabnormally high concentrations of hemoglobin, bilirubin, and/ortriglyceride. Abnormally high concentrations of these interferingsubstances can be caused by a pathological state of the patient or elseby inappropriate sample acquisition or storage.

SUMMARY

According to various embodiments, a method for determining the activityof proteolytic coagulation factors in a sample can be provided which isless susceptible to interference by disruptive substances intrinsic tothe sample.

According to an embodiment, a method for determining the activity of aproteolytic coagulation factor in a sample may comprise the followingsteps:

-   -   a. providing and incubating a reaction mixture comprising        -   i. the sample,        -   ii. an agent for direct or indirect activation of the            proteolytic coagulation factor in the sample,        -   iii. a cleavable substrate which has at least one cleavage            site for the activated coagulation factor,        -   iv. a solid phase to which the cleavable substrate is bound            or becomes bound during the incubation;    -   b. separating off the solid phase; and    -   c. determining the amount of solid-phase-bound, uncleaved        substrate.

According to a further embodiment, the cleavable substrate can bepresent in a separate reagent which is added to the reaction mixture,and wherein the reaction mixture may comprise a solid phase to which thecleavable substrate becomes bound during the incubation. According to afurther embodiment, the cleavable substrate may comprise a peptide whichconsists of 3 to 150 amino acid residues. According to a furtherembodiment, the cleavable substrate can be a natural substrate for theactivated coagulation factor and a constituent of the sample, andwherein the reaction mixture comprises a solid phase to which thecleavable substrate becomes bound during the incubation. According to afurther embodiment, the reaction mixture may comprise a solid phase towhich the cleavable substrate is covalently bonded. According to afurther embodiment, the cleavable substrate may have a first bindingpartner A of a first binding pair A/B, and wherein the solid phase hasthe second binding partner B of the first binding pair A/B, and whereinthe cleavable substrate is bound to the solid phase by the binding ofbinding partners A and B or becomes bound during the incubation.According to a further embodiment, the binding partners A and B can bechosen such that they form a binding pair A/B selected from the groupconsisting of FLAG-tag/anti-FLAG-tag antibody, His-tag/anti-His-tagantibody, fluorescein/anti-fluorescein antibody. According to a furtherembodiment, the cleavable substrate may have a first binding partner Xof a second binding pair X/Y, and wherein the second binding partner Yof the second binding pair X/Y is associated with a component of asignal-producing system. According to a further embodiment, the bindingpartners X and Y can be chosen such that they form a binding pair X/Yselected from the group consisting of biotin/avidin,biotin/streptavidin. According to a further embodiment, an agentselected from the group consisting of thromboplastin, factor IIa, factorVIIa, factor IXa, factor Xa, factor XIa, factor XIIa, activated proteinC, snake poisons, negatively charged phospholipids, calcium ions, tissuefactor, silica, kaolin, ellagic acid, and celite may be used for director indirect activation of the proteolytic coagulation factor. Accordingto a further embodiment, an inhibitor of fibrin aggregation may furtherbe added to the reaction mixture.

According to another embodiment, the above described method may be usedfor determining the activity of a proteolytic coagulation factorselected from the group consisting of factor II, factor VII, factor IX,factor X, factor XI, factor XII, and protein C.

According to yet another embodiment, a method for quantitativelydetermining an anticoagulant in a sample may comprises the followingsteps:

-   -   a. providing and incubating a reaction mixture comprising        -   i. the sample,        -   ii. a defined amount of an activated coagulation factor            whose activity is directly or indirectly influenceable by            the anticoagulant to be determined, wherein the activated            coagulation factor is present in a separate reagent which is            added to the reaction mixture,        -   iii. a cleavable substrate which has at least one cleavage            site for the activated coagulation factor,        -   iv. a solid phase to which the cleavable substrate is bound            or becomes bound during the incubation;    -   b. separating the solid phase; and    -   c. determining the amount of solid-phase-bound, uncleaved        substrate.

According to a further embodiment, the above method may be used forquantitatively determining an anticoagulant selected from the groupconsisting of LMW heparin, HMW heparin, heparinoids, direct thrombininhibitors, and direct factor Xa inhibitors. According to a furtherembodiment, the reaction mixture may comprise a defined amount of anactivated coagulation factor selected from the group consisting offactor IIa and factor Xa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the absorbance values at 450 nm of samples in which theextrinsic pathway of coagulation was activated and in which thrombinactivity was determined,

FIG. 2 shows the absorbance values at 450 nm of samples, havingdifferent concentrations of Refludan®

DETAILED DESCRIPTION

As stated above, a method may comprise the following steps:

-   -   a. providing and incubating a reaction mixture comprising        -   i. the sample,        -   ii. an agent for direct or indirect activation of the            proteolytic coagulation factor in the sample,        -   iii. a cleavable substrate which has at least one cleavage            site for the activated coagulation factor,        -   iv. a solid phase to which the cleavable substrate is bound            or becomes bound during the incubation;    -   b. separating off the solid phase; and    -   c. determining the amount of solid-phase-bound, uncleaved        substrate.

The amount of uncleaved substrate bound to the solid phase is inverselyproportional to the activity of the proteolytic coagulation factor to bedetermined.

Providing the reaction mixture always comprises contacting the sample,preferably a blood or plasma sample, with an agent for direct orindirect activation of the proteolytic coagulation factor to bedetermined in the sample and with a solid phase. The cleavablesubstrate, which has at least one cleavage site for the activatedcoagulation factor, can be added to the reaction mixture in differentways:

-   -   The cleavable substrate, for example a synthetic peptide or a        purified protein which is not bound to a solid phase, can be        present in a separate reagent which is added to the reaction        mixture. The reaction mixture then comprises a solid phase to        which the cleavable substrate becomes bound during the        incubation.    -   The cleavable substrate, for example a synthetic peptide or a        purified protein, is already bound to the solid phase and is,        together with the solid phase as a constituent thereof,        contacted with the sample and the agent for activation of the        proteolytic coagulation factor.    -   The cleavable substrate is a natural substrate for the activated        coagulation factor and naturally occurring in the sample and is,        as a result, added to the reaction mixture with the sample as a        constituent of the sample material. The reaction mixture then        comprises a solid phase to which the cleavable natural substrate        becomes bound during the incubation.

Cleavable substrates which have at least one cleavage site for anactivated coagulation factor are well known to a person skilled in theart. A cleavable substrate can be a synthetically, recombinantly, orbiotechnologically produced molecule or a natural molecule which isbroken up into two cleavage products through the action of the activatedcoagulation factor. A cleavable substrate can consist wholly or partlyof a peptide. Preferably, it may comprise a peptide portion at least inthe region of the cleavage site. Preferably, the peptide portion of acleavable substrate may consist of 3 to about 150 amino acid residues.

In another embodiment, the cleavable substrate can consist of a completeprotein or of a protein fragment. A cleavable substrate can also be anatural substrate of an activated coagulation factor. An example of anatural cleavable substrate is factor V, which has cleavage sites foractivated protein C, factor Xa, factor IIa (thrombin), and plasmin. Afurther example is fibrinogen, which has cleavage sites for factor IIa(thrombin). Yet a further example is factor II (prothrombin), which hascleavage sites for factor IIa (thrombin), factor Xa, and various snakepoisons, such as, for example, ecarin or textarin.

In an embodiment of the method, the cleavable substrate, which has atleast one cleavage site for the activated coagulation factor, is boundto a solid phase.

The term “bound” is to be understood broadly and comprises, for example,covalent bonding and noncovalent binding, direct and indirect binding,adsorption to a surface, and inclusion in an indentation or a cavity,etc. In the case of covalent bonding, the cleavable substrate is boundto the solid phase via a chemical bond. Examples of noncovalent bindingare surface adsorption, inclusion in cavities, or the binding of twospecific binding partners. In addition to direct binding to the solidphase, the cleavage substrate can also be bound indirectly to the solidphase via a specific interaction with other specific binding partners.

In an embodiment, the cleavable substrate has a first binding partner Aof a first binding pair A/B, and the solid phase has the binding partnerB, and the substrate is bound to the solid phase by the binding ofbinding partners A and B.

In another embodiment of the method, the cleavable substrate, which hasat least one cleavage site for the activated coagulation factor, becomesbound to the solid phase during the incubation of the reaction mixture.For this purpose, the cleavable substrate has a first binding partner Aof a first binding pair A/B, and the solid phase has the binding partnerB, and the substrate becomes bound to the solid phase by the binding ofbinding partners A and B during the incubation of the reaction mixture.

Suitable binding pairs A/B are, in particular, antigen/antibodycombinations, wherein the binding partner A is an antigenic epitope ofthe cleavable substrate. The antigenic epitope can be a natural linearor conformational epitope of a natural protein or protein fragment, moreparticularly when a natural substrate present in the sample is used as acleavable substrate. The antigenic epitope can also be a heterologouslinear or conformational epitope of a modified cleavable substrate.Examples of heterologous linear or conformational epitopes are FLAG- orHis- or fluorescein tags which are used more particularly for labelingpeptides or proteins. The solid-phase-bound binding partner B must bechosen such that the cleavable substrate can become specifically bound.Preferably, the binding partner B may consist of an antibody or anantigen-binding fragment thereof. Particularly preferred binding pairsA/B may be FLAG-tag/anti-FLAG-tag antibody, His-tag/anti-His-tagantibody, and fluorescein/anti-fluorescein antibody.

In a further embodiment of the method, the cleavable substrate has afirst binding partner X of a second binding pair X/Y which interactswith the second binding partner Y of the second binding pair X/Y,wherein the second binding partner Y is associated with a component of asignal-producing system. In this way, uncleaved substrate can bedetected. The binding partner X is arranged in a substrate region which,provided the substrate is cleaved by the activated proteolyticcoagulation factor, is separated from the solid-phase-bound region ofthe substrate.

Suitable binding pairs X/Y are, for example, antigen/antibodycombinations, wherein the binding partner X is an antigenic epitope ofthe cleavable substrate. The antigenic epitope can be a natural linearor conformational epitope of a natural protein or protein fragment, moreparticularly when a natural substrate present in the sample is used as acleavable substrate. The antigenic epitope can also be a heterologouslinear or conformational epitope of a modified cleavable substrate.Examples of heterologous linear or conformational epitopes are FLAG- orHis- or fluorescein tags which are used more particularly for labelingpeptides or proteins. Further suitable binding pairs X/Y are, forexample, biotin/avidin and biotin/streptavidin.

The second binding partner Y is associated with a component of asignal-producing system.

A “signal-producing system” can be one or more components, wherein atleast one component is a detectable label. A label is to be understoodto mean any molecule which itself produces a signal or which can inducethe production of a signal, such as, for example, a fluorescentsubstance, a radioactive substance, an enzyme, or a chemiluminescentsubstance. The signal can, for example, be detected or measured by meansof enzyme activity, luminescence, light absorbance, light scattering,emitted electromagnetic or radioactive radiation, or chemical reaction.

A label is itself capable of generating a detectable signal so that nofurther components are necessary. Many organic molecules absorbultraviolet and visible light, whereby these molecules can enter anexcited energy state and release the absorbed energy in the form oflight of a wavelength other than that of the excitation light. Yet otherlabels, such as, for example, radioactive isotopes or dyes, can generatea detectable signal directly.

Yet other labels require further components for signal generation, i.e.,the signal-producing system in such a case includes all the componentsrequired for signal production, such as, for example, substrates,coenzymes, quenchers, accelerants, additional enzymes, substances whichreact with enzyme products, catalysts, activators, cofactors,inhibitors, ions, etc.

Suitable labels are, for example, enzymes including horseradishperoxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase,alcohol dehydrogenase, glucose oxidase, β-galactosidase, luciferase,urease, and acetylcholinesterase; dyes; fluorescent substances includingfluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,ethidium bromide, 5-dimethylaminonaphthalene-1-sulfonyl chloride, andfluorescent chelates of rare earths; chemiluminescent substancesincluding luminol, isoluminol, acridinium compounds, olefin, enolethers, enamine, aryl vinyl ethers, dioxene, arylimidazole, lucigenin,luciferin, and aequorin; sensitizers including eosin,9,10-dibromoanthracene, methylene blue, porphyrin, phthalocyanine,chlorophyll, Rose Bengal; coenzymes; enzyme substrates; radioactiveisotopes including ¹²⁵I, ¹³¹I, ¹⁴C, ³H, ³²P, ³³P, ³⁵S, ⁵¹Cr, ⁵⁹Fe, ⁵⁷Co,and ⁷⁵Se.

The solid phase to which the cleavable substrate is bound or becomesbound during the incubation comprises an article which consists ofporous and/or nonporous, generally water-insoluble material and whichcan have a wide variety of different forms, such as, for example, thoseof vessels, small tubes, microtiter plates, beads, microparticles, rods,strips, filter or chromatography paper, etc. Generally, the surface ofthe solid phase is hydrophilic or can be made hydrophilic. The solidphase can consist of a wide variety of different materials, such as, forexample, inorganic and/or organic materials, synthetic materials,naturally occurring materials and/or modified naturally occurringmaterials. Examples of solid phase materials are polymers, such as, forexample, cellulose, nitrocellulose, cellulose acetate, polyvinylchloride, polyacrylamide, crosslinked dextran molecules, agarose,polystyrene, polyethylene, polypropylene, polymethacrylate, or nylon;ceramic, glass, metals, more particularly noble metals such as gold andsilver; magnetite; mixtures or combinations thereof; etc.

The solid phase can have a coating composed of one or more layers, forexample, of proteins, carbohydrates, lipophilic substances, biopolymers,organic polymers, or mixtures thereof, in order, for example, todiminish or to prevent the nonspecific binding of sample constituents tothe solid phase or, for example, to achieve improvements with regard tosuspension stability of particulate solid phases, storage stability,shape-imparting stability, or resistance to UV light, microbes, or otherdestructively acting agents.

To directly or indirectly activate the proteolytic coagulation factor inthe sample, the sample is usually mixed with an agent which causesdirect or indirect activation of the proteolytic coagulation factor.Direct activation is to be understood to mean use of an agent whichdirectly activates the proteolytic coagulation factor to be determined,irrespective of the presence of other coagulation factors. Indirectactivation is to be understood to mean use of an agent which activatesone or more blood coagulation factors of the blood coagulation cascade,which in turn activate the proteolytic coagulation factor to beexamined. The type of agent depends on which coagulation factor is to bedetermined, on whether the activity of the coagulation factor alone isto be determined, or on whether the functionality of the bloodcoagulation cascade or of a subdomain of the blood coagulation cascade(extrinsic or intrinsic pathway) is to be determined with the aid of acoagulation factor. Substances and specific mixtures of varioussubstances which make direct or indirect activation of proteolyticcoagulation factors possible are well known to a person skilled in theart and comprise, for example, phospholipids, such as, for example,negatively charged phospholipids; lipoproteins, such as, for example,thromboplastin; proteins, such as, for example, tissue factor, activatedserine proteases, such as, for example, factor IIa (thrombin), factorVIIa, factor IXa, factor Xa, factor XIa, factor XIIa, or activatedprotein C; snake poisons, such as, for example, PROTAC® enzyme, ecarin,textarin, noscarin, batroxobin, thrombocytin, or Russells's viper venom(RVV); contact activators, such as, for example, silica, kaolin, ellagicacid, or celite. Further substances which may comprise an agent are, forexample, buffer substances, salts, detergents, ions, more particularlycalcium ions and chelating agents.

A “sample” is to be understood to mean, for the purpose of theinvention, the material which presumably contains the proteolyticcoagulation factor to be detected. The term sample comprises, moreparticularly, human or animal body fluids, chiefly blood and plasma.

After provision of the reaction mixture, which comprises the sample, anagent for activation of the proteolytic coagulation factor, a cleavablesubstrate, and a solid phase to which the cleavable substrate is boundor becomes bound, the reaction mixture is incubated for a certain lengthof time in order to ensure sufficient activation of the coagulationfactor, sufficient cleavage of the substrate by the activatedcoagulation factor, and, if appropriate, sufficient binding of thecleavable substrate or the cleavage product of the substrate to thesolid phase. The term “sufficient” is to be understood to mean such thatthe method as a whole makes quantitative determination of the activityof the coagulation factor possible. The optimal duration of incubationof a certain test setup can be ascertained experimentally.

In an embodiment, an inhibitor of fibrin aggregation can further beadded to the reaction mixture. A fibrin aggregation inhibitor is to beunderstood to mean a substance, more particularly a syntheticoligopeptide, which inhibits the joining together (polymerization) offibrin monomers which arise through the action of thrombin and thusprevents clot formation in the reaction mixture (see, for example, EP 0456 152 B1).

After the reaction mixture has been incubated, the solid phase, and thusthe constituents bound to it, is separated from the rest of theconstituents of the reaction mixture. Separation can be carried out indifferent ways, depending on the type of solid phase, for example, bycentrifugation, filtration, magnetic separation, or by aspiration of theliquid phase of the reaction mixture. After removal of the solid phase,at least one wash step can be carried out in order to remove residualreaction mixture from the solid phase as completely as possible and/orto prepare the solid phase for the subsequent detection reaction. Forthis purpose, the solid phase is incubated with a wash solution,preferably with a buffer solution, and subsequently separated again fromthe wash solution.

Determining the amount of solid-phase-bound, uncleaved substrate can becarried out in different ways, depending on the type of detection systemused, for example, by incubation of the solid phase with a reagent whichcomprises substances which interact specifically with the uncleavedsubstrate and generate a measurable signal. Preferably, determining theamount of solid-phase-bound, uncleaved substrate may be carried out byincubating the solid phase with a detection reagent which comprises thebinding partner Y of the second binding pair X/Y, in which case bindingpartner Y binds specifically to the binding partner X of the uncleavedsubstrate. The binding partner Y can either be associated directly witha signal-giving component or become associated with a signal-givingcomponent.

The amount or strength of the signal is proportional to the amount ofsolid-phase-bound, uncleaved substrate and thus inversely proportionalto the activity of the proteolytic coagulation factor.

The method according to various embodiments for determining aproteolytic coagulation factor is particularly suitable for determiningthe proteolytic coagulation factors factor II, factor VII, factor IX,factor X, factor XI, factor XII, or protein C.

According to other embodiments, a method for quantitatively determiningan anticoagulant in a sample can be provided which is less vulnerable todisruptive substances intrinsic to the sample.

According to these other embodiments, a method may comprise thefollowing steps:

-   -   a. providing and incubating a reaction mixture comprising        -   i. the sample,        -   ii. a defined amount of an activated coagulation factor            whose activity is directly or indirectly influenceable by            the anticoagulant to be determined, wherein the activated            coagulation factor is present in a separate reagent which is            added to the reaction mixture,        -   iii. a cleavable substrate which has at least one cleavage            site for the activated coagulation factor,        -   iv. a solid phase to which the cleavable substrate is bound            or becomes bound during the incubation;    -   b. separating off the solid phase; and    -   c. determining the amount of solid-phase-bound, uncleaved        substrate.

In this method, a known, defined amount of an activated coagulationfactor is added to the reaction mixture. Which activated coagulationfactor is added depends on which anticoagulant is to be determined.

To determine a heparin, i.e., a high-molecular-weight, unfractionatedheparin (HMW heparin) or a low-molecular-weight heparin (LMW heparin) ora heparinoid, the addition of factor IIa (thrombin) or of factor Xa isparticularly suitable. To determine a direct thrombin inhibitor, forexample, argatroban, melagatran, ximelagatran, bivalirudin, dabigatran,or hirudin, the addition of factor IIa (thrombin) is particularlysuitable. To determine a direct factor Xa inhibitor, for example,rivaroxaban, the addition of factor Xa is particularly suitable.

The more anticoagulant that is present in the sample, the stronger isthe inhibition of the activated coagulation factor that has been addedand the less cleavable substrate that is cleaved. The amount ofsolid-phase-bound, uncleaved substrate is therefore proportional to theamount or activity, present in the sample, of the anticoagulant to bedetermined.

Apart from the fact that, in the method according to various embodimentsfor quantitatively determining an anticoagulant,

-   -   use is made of a defined amount of an activated coagulation        factor whose activity is directly or indirectly influenceable by        the anticoagulant to be determined, wherein the activated        coagulation factor is present in a separate reagent which is        added to the reaction mixture, and that    -   therefore the reaction mixture need not contain an agent for        activating a proteolytic coagulation factor,        the above explanations for carrying out the method for        determining a proteolytic coagulation factor also apply to the        method according to various embodiments for quantitatively        determining an anticoagulant.

The following examples serve to illustrate the present invention and arenot to be understood as limiting.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the absorbance values at 450 nm of samples in which theextrinsic pathway of coagulation was activated by Innovin® (athromboplastin reagent) and in which thrombin activity was determined(see example 1). The samples are standard human plasma (SHP), whichfully comprises all coagulation factors, and also plasmas deficient infactors of the extrinsic coagulation pathway (F II, F V, F VII, F X) orof the intrinsic coagulation pathway (F VIII, F IX, F XI). Depending onthe amount of thrombin in the activated sample, which is in turndependent on the presence of the factors of the extrinsic coagulationpathway, the thrombin-sensitive peptide substrate is cleaved. The morethrombin that is present in the sample, the more thrombin substrate thatis cleaved and the less uncleaved thrombin substrate that can bedetected. The measured absorbance values are thus inversely proportionalto the thrombin activity in the sample, i.e., to the activity of theextrinsic coagulation cascade. In the experiment shown, all plasmasdeficient in factors of the extrinsic coagulation pathway (moreparticularly the F II-deficient plasma) therefore have higher absorbancevalues than normal plasma (SHP), whereas plasma lacking factors notinvolved in the extrinsic coagulation pathway (factor VIII, IX, XI) showno differences in signal compared to SHP. Therefore, using the testshown, it was possible to clearly detect a deficiency in factors of theextrinsic coagulation pathway in plasmas.

FIG. 2 shows the absorbance values at 450 nm of samples, havingdifferent concentrations of Refludan®, to which defined amounts ofthrombin were added (thr. 1 IU/ml, thr. 10 IU/ml) and in which thrombinactivity was determined (see example 2). The more of the direct thrombininhibitor Refludan® that is present in a sample, the more of the addedthrombin that is inhibited, the less thrombin substrate that is cleaved,and the more uncleaved thrombin substrate that can be detected. Themeasured absorbance values are thus proportional to thethrombin-inhibiting activity of the Refludan® in the sample. It wasevident that the sensitivity of the method is particularly good forsamples having therapeutically low Refludan® concentrations (to 1 μg/ml)upon addition of a comparatively low amount of thrombin (1 IU/ml), whilethe sensitivity of the method is particularly good for samples havingtherapeutically high Refludan® concentrations (1-5 μg/ml) upon additionof a comparatively high amount of thrombin (10 IU/ml).

EXAMPLES Example 1 Method for Determining Thrombin Activity

First, microtiter plates (Nunc-Thermo Fisher, Roskilde, Denmark) werecoated with a monoclonal FLAG-epitope-specific antibody (MAK M2, SigmaAldrich, Munich, Germany). Subsequently, a thrombin-specific peptidesubstrate comprising seven amino acid residues, specifically leucine,valine, proline, arginine, glycine, phenylalanine, glycine in saidsequence, which has a FLAG-tag epitope at the amino-terminal end, andwhich is biotinylated at the carboxy-terminal end was incubated with thesolid-phase-bound anti-FLAG antibody and thus bound to the solid phase.To each well, the following reagents were subsequently added in thefollowing order:

25 μl of a fibrin aggregation inhibitor solution (6 mg/ml of a syntheticoligopeptide having the amino acid residues glycine, proline, arginine,proline, alanine in said sequence); 25 μl of sample; and 25 μl ofInnovin® (recombinant human tissue factor having syntheticphospholipids, Siemens Healthcare Diagnostics, Marburg, Germany) as acoagulation activator.

Standard human plasma (SHP) and plasmas deficient in coagulation factor(factor II, factor X, factor V, factor VII, factor VIII, factor IX,factor XI) were used as samples.

The reaction mixture was commixed and incubated at 37° C. for 15minutes. The reaction mixture was then aspirated, and each well waswashed three times with, in each case, 250 μl of wash buffer. To detectuncleaved, solid-phase-bound thrombin substrate, 100 μl of astreptavidin/peroxidase (POD) conjugate solution (0.33 μg/ml, SigmaAldrich) were added to each well and in turn incubated at 20-25° C. for30 minutes. The conjugate solution was then aspirated, and each well waswashed three times with, in each case, 250 μl of wash buffer.Subsequently, 100 μl of a buffer solution comprising 0.5 g/L TMB(3,3′,5,5′-tetramethylbenzidine dihydrochloride) and 0.1 g/L hydrogenperoxide were added to each well and in turn incubated at 20-25° C. for30 minutes. To terminate the peroxidase reaction, 100 μl of 0.5 Nsulfuric acid were added to each well, and the absorbance at 450 nmminus the absorbance at reference wavelength 650 nm was determined bymeans of Sunrise® MTP photometer (Tecan Trading AG, Switzerland).

The results are displayed in FIG. 1.

Example 2 Method for Quantitatively Determining the Direct ThrombinInhibitor Refludan®

As described in example 1, a thrombin-specific peptide substrate,comprising seven amino acid residues, which has a FLAG-tag epitope atthe amino-terminal end and is biotinylated at the carboxy-terminal endwas bound to the solid phase (microtiter plate) via an anti-FLAGantibody.

To each well, the following reagents were added in the following order:

-   50 μl of a fibrin aggregation inhibitor solution (3 mg/ml of a    synthetic oligopeptide having the amino acid residues glycine,    proline, arginine, proline, alanine in said sequence); 25 μl of    sample; and-   25 μl of bovine α-thrombin solution (4 or 40 IU of bovine α-thrombin    per ml, 10 KIE/ml aprotinin, 150 mM/L NaCl, 5 mg/ml bovine serum    albumin, 10 mg/ml mannitol, 5 μg/ml hexadimethrine bromide).

Through the different thrombin concentrations, sensitivity in varioustherapeutic areas can be ensured.

Standard human plasma (SHP) and normal human citrate plasma samples wereused as samples. These samples were aliquoted, and Refludan® (lepirudin,recombinant hirudin, CSL Behring GmbH, Marburg, Germany) was added toeach aliquot to give final concentrations of 0.0 μg/ml, 0.2 μg/ml, 1.0μg/ml, and 5 μg/ml.

The detection of uncleaved, solid-phase-bound thrombin substrate wascarried out as described in example 1, and the absorbance at 450 nmminus the absorbance at reference wavelength 650 nm was determined bymeans of Sunrise® MTP photometer (Tecan Trading AG, Switzerland).

The results are displayed in FIG. 2.

What is claimed is:
 1. A method for quantitatively analyzing ananticoagulant in a sample, wherein the method comprises the followingsteps: a. providing and incubating a reaction mixture comprising i. thesample, ii. a defined amount of an activated coagulation factor whoseactivity is directly or indirectly influenceable by the anticoagulant tobe determined, wherein the activated coagulation factor is present in aseparate reagent which is added to the reaction mixture, iii. acleavable substrate which has at least one cleavage site for theactivated coagulation factor, iv. a solid phase to which the cleavablesubstrate is bound or becomes bound during the incubation; b. separatingthe solid phase; and c. determining the amount of solid-phase-bound,uncleaved substrate, wherein the determined amount of thesolid-phase-bound uncleaved substrate indicates a quantitative measureof the anticoagulant in the sample.
 2. The method as claimed in claim 1,wherein the cleavable substrate is present in a separate reagent whichis added to the reaction mixture, and wherein the reaction mixturecomprises a solid phase to which the cleavable substrate becomes boundduring the incubation.
 3. The method as claimed in claim 2, wherein thecleavable substrate comprises a peptide which consists of 3 to 150 aminoacid residues.
 4. The method as claimed in claim 1, wherein thecleavable substrate is a natural substrate for the activated coagulationfactor and a constituent of the sample, and wherein the reaction mixturecomprises a solid phase to which the cleavable substrate becomes boundduring the incubation.
 5. The method as claimed in claim 1, wherein thereaction mixture comprises a solid phase to which the cleavablesubstrate is covalently bonded.
 6. The method as claimed in claim 1,wherein the cleavable substrate has a first binding partner A of a firstbinding pair A/B, and wherein the solid phase has the second bindingpartner B of the first binding pair A/B, and wherein the cleavablesubstrate is bound to the solid phase by the binding of binding partnersA and B or becomes bound during the incubation.
 7. The method as claimedin claim 6, wherein the binding partners A and B are chosen such thatthey form a binding pair A/B selected from the group consisting ofFLAG-tag/anti-FLAG-tag antibody, His-tag/anti-His-tag antibody,fluorescein/anti-fluorescein antibody.
 8. The method as claimed in claim1, wherein the cleavable substrate has a first binding partner X of asecond binding pair X/Y, and wherein the second binding partner Y of thesecond binding pair X/Y is associated with a component of asignal-producing system.
 9. The method as claimed in claim 8, whereinthe binding partners X and Y are chosen such that they form a bindingpair X/Y selected from the group consisting of biotin/avidin,biotin/streptavidin.
 10. The method as claimed in claim 1, wherein aninhibitor of fibrin aggregation is further added to the reactionmixture.
 11. The method as claimed in claim 1, wherein the step ofseparating the solid phase is performed by centrifugation, filtration,magnetic separation, or by aspiration of the liquid phase of thereaction mixture.
 12. The method as claimed in claim 1, wherein at leastone wash step is carried out after separating the solid phase.
 13. Themethod as claimed in claim 12, wherein the solid phase is incubated witha wash solution and subsequently separated again from the wash solution.14. The method as claimed in claim 13, wherein the wash solution is abuffer solution.
 15. The method as claimed in claim 1 for quantitativelydetermining an anticoagulant selected from the group consisting of LMWheparin, HMW heparin, heparinoids, direct thrombin inhibitors, anddirect factor Xa inhibitors.
 16. The method as claimed in claim 1,wherein the reaction mixture comprises a defined amount of an activatedcoagulation factor selected from the group consisting of factor IIa andfactor Xa.
 17. The method as claimed in claim 1, wherein the determinedamount of the solid-phase-bound uncleaved substrate is proportional to aquantitative measure of an amount or activity of the anticoagulant inthe sample.